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Comparison of Human and Drosophila Atlastin Gtpases

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Comparison of Human and Drosophila Atlastin Gtpases Protein Cell 2015, 6(2):139–146 DOI 10.1007/s13238-014-0118-0 Protein & Cell RESEARCH ARTICLE Comparison of human and Drosophila atlastin GTPases Fuyun Wu1,2, Xiaoyu Hu1,2, Xin Bian1,2, Xinqi Liu3,4, Junjie Hu1,2,5& 1 Department of Genetics and Cell Biology, College of Life Sciences, Nankai University, Tianjin 300071, China 2 Tianjin Key Laboratory of Protein Sciences, Tianjin 300071, China 3 Department of Biochemistry and Molecular Biology, College of Life Sciences, Nankai University, Tianjin 300071, China 4 State Key Laboratory of Medicinal Chemical Biology, Tianjin 300071, China 5 National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China & Correspondence: [email protected] (J. Hu) Received October 9, 2014 Accepted October 27, 2014 Cell & ABSTRACT KEYWORDS endoplasmic reticulum, membrane fusion, atlastin, GTPase, X-ray crystallography Formation of the endoplasmic reticulum (ER) network requires homotypic membrane fusion, which involves a Protein class of atlastin (ATL) GTPases. Purified Drosophila ATL INTRODUCTION is capable of mediating vesicle fusion in vitro, but such activity has not been reported for any other ATLs. Here, In eukaryotic cells, the endoplasmic reticulum (ER) is com- we determined the preliminary crystal structure of the posed of interconnected tubules and sheets (Shibata et al., cytosolic segment of Drosophila ATL in a GDP-bound 2006). The merging of ER membranes, i.e. homotypic state. The structure reveals a GTPase domain dimer with fusion, is required for ER network formation and mediated by the subsequent three-helix bundles associating with a dynamin-like membrane bound GTPase family known as their own GTPase domains and pointing in opposite atlastins (ATLs) (Hu et al., 2009; Orso et al., 2009). No ATL directions. This conformation is similar to that of human has been found in yeast and plant cells, but a similar class of ATL1, to which GDP and high concentrations of inor- GTPases, named Sey1p or RHD3, plays an analogous role ganic phosphate, but not GDP only, were included. in ER morphogenesis (Hu et al., 2009; Anwar et al., 2012; Drosophila ATL restored ER morphology defects in Zhang et al., 2013). Depletion of Drosophila or zebrafish ATL mammalian cells lacking ATLs, and measurements of results in neuronal defects (Lee et al., 2008; Lee et al., 2009; nucleotide-dependent dimerization and GTPase activity Fassier et al., 2010). Deletion or mutation of RHD3 leads to were comparable for Drosophila ATL and human ATL1. defects in plant growth and short and wavy root hairs However, purified and reconstituted human ATL1 (Schiefelbein and Somerville, 1990; Wang et al., 1997; exhibited no in vitro fusion activity. When the cytosolic Stefano et al., 2012). Depletion or mutation of human ATL1, segment of human ATL1 was connected to the trans- a dominant form in the central nervous system, causes membrane (TM) region and C-terminal tail (CT) of Dro- unbranched ER at the cellular level (Hu et al., 2009) and sophila ATL, the chimera still exhibited no fusion hereditary spastic paraplegia (HSP), a neurodegenerative activity, though its GTPase activity was normal. These disease characterized by axon shortening in corticospinal results suggest that GDP-bound ATLs may adopt mul- motor neurons and progressive spasticity and weakness of tiple conformations and the in vitro fusion activity of ATL the lower limbs, at the organism level (Zhao et al., 2001; cannot be achieved by a simple collection of functional Salinas et al., 2008). domains. Several assays have been developed to study the fuso- genic activity of these GTPases. First, yeast cells lacking Sey1p and either Yop1p or Rtn1p, members of the ER tubule- forming protein family, exhibit abnormal ER morphology (Hu et al., 2009). Restoration of the ER network can then be Fuyuan Wu and Xiaoyu Hu contributed equally to this work. monitored upon expression of any ER fusogen candidates © The Author(s) 2014. This article is published with open access at Springerlink.com and journal.hep.com.cn RESEARCH ARTICLE Fuyun Wu et al. A GTPase 3HBTMsCT C dmATL GTPase-1 GTPase-2 hsATL1 B Form 1 Form 2 Form 3 3HB-1 3HB-2 +GDP + - +GDP +GDP/Pi GDP/AlF 4 +GppNp D E hsATL1 + GDP/Pi hsATL1 + GDP/Pi Cell & dmATL + GDP dmATL + GDP Figure 1. Crystal structure of the cytosolic domain of Drosophila ATL. (A) Schematic diagrams of Drosophila ATL and human ATL1. (B) Human ATL1 dimers observed in crystal form 1 (PDB code: 3QNU, left, GDP-bound), form 2 (PDB code: 3QOF, middle, Protein GDP-bound in the presence of inorganic phosphate), and form 3 (PDB code: 4IDO, right, GDP/AlF4-bound or GppNp-bound) are shown. The protomers in the dimer are shown in green and purple cartoon representation. (C) Structure of the GDP-bound form of Drosophila ATL. The protomers in the dimer are shown in green and purple cartoon representation. GDP is shown in orange stick representation, and magnesium ion is shown as a yellow sphere. 3HB, three-helix bundle. (D) Structural comparison of human and Drosophila ATLs. Human ATL1 (hsATL1) is shown in pink and Drosophila ATL (dmATL) is shown in green. GDP in dmATL is shown in orange, and magnesium ion is shown in yellow. (E) As in C, but with a dimer. Superposition was performed using the protomers on the left. (Anwar et al., 2012; Zhang et al., 2013). Second, ER fusion facing each other, but the position of the 3HB differs can be followed during mating of haploid yeast cells carrying (Fig. 1B). In form 1, ATL1 is in complex with GDP, the two cytosolic and ER markers (Anwar et al., 2012). Deletion of 3HBs associate with the paired GTPase domains and form a Sey1p significantly reduces the fusion process, whereas crossover conformation. In this case, the connecting TMs expression of a functional ER fusogen, such as human ATL1 would have to sit in the same membranes; thus, the structure or RHD3 (Anwar et al., 2012; Zhang et al., 2013), can rescue corresponds to a “post-fusion” state in which the membranes this defect. Moreover, purified drosophila ATL has been have already fused. In form 2, ATL1 is also GDP-bound, but reconstituted into proteoliposomes, and signals of lipid mixing in the presence of high concentration of inorganic phos- in the presence of magnesium and GTP provide direct evi- phate. The two 3HBs associate with their own GTPase dence for membrane fusion mediated by ATL (Orso et al., domains and point in opposite directions. This structure 2009; Bian et al., 2011). Using this assay, Sey1p and mem- implies that two ATL molecules likely sit in apposing mem- bers of the RHD3 family have been confirmed to mediate branes, corresponding to a membrane-tethered “pre-fusion” fusion in vitro. However, drosophila ATL is the only ATL that state. In form 3, ATL is crystallized with either GDP/AlF4 or has been reported to have fusogenic activity in vitro. GppNp. The two 3HBs come even closer than in form 1. ATL contains an N-terminal GTPase and a three-helix Collectively, ATL-mediated fusion requires dimerization bundle (3HB), followed by two closely spaced transmem- resulting from GTP binding and conformational changes brane (TM) segments and a C-terminal tail (CT) (Fig. 1A). In induced by GTP hydrolysis (Bian et al., 2011; Byrnes and an effort to understand the ATL-associated fusion mecha- Sondermann, 2011; Hu et al., 2011; Lin et al., 2012) nisms, three crystal structures of the N-terminal cytosolic In addition to the N-terminal cytosolic domain, the TM and domain of human ATL1 have been determined (Bian et al., CT have also been shown to play important roles in fusion 2011; Byrnes and Sondermann, 2011). In all three forms of (Liu et al., 2012). The TM of ATL appears to be more than a ATL, the molecule forms a dimer with the GTPase domains membrane anchor, as deletion or replacement of the TM 140 © The Author(s) 2014. This article is published with open access at Springerlink.com and journal.hep.com.cn Comparing human and Drosophila ATL RESEARCH ARTICLE region results in a loss of fusion activity. The CT of ATL forms Table 1. Data collection and refinement statistics an amphipathic helix that binds and destabilizes the mem- branes to facilitate fusion. A synthetic peptide corresponding Data collection to parts of the CT of Drosophila ATL was able to restore Space group C121 fusion defects in the tailless ATL mutant. The importance of Wavelength (Å) 1.54 ATL-CT is consistent with the fact that a lack of CT in human Unit cell ATL1 mutants causes HSP (Liu et al., 2012). Drosophila ATL has been used extensively to study a, b, c (Å) 71.62, 63.91, 107.76 membrane fusion in vitro, but structural information about α, β, γ (°) 90, 101.6, 90 ATL has been obtained purely from human ATL1. Whether Molecules/ASU 1 human ATL can mediate fusion in vitro is also not clear. Resolution range (Å)a 50–2.87 (2.92–2.87) Here, we determined the structure of the N-terminal cytosolic a domain of Drosophila ATL and compared fusogenic features Completeness (%) 91.93 (80.49) of human and Drosophila ATL. Redundancya 3.1 (2.5) No. of total reflections 31354 No. of unique reflections 10190 RESULTS I/σa 26.4 (3.8) Crystal structure of the cytosolic domain of Drosophila Rsym (%)a,b 8.1 (31.5) ATL Refinement statistics Cell To gain insight into Drosophila ATL-mediated membrane Resolution (Å) 2.87 & fusion, we determined the crystal structure of cytATL. Resi- No. of reflections 10185 dues 1–422 were expressed in E. coli, purified, and crys- c tallized in the presence of GDP.
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